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School of Biological Sciences

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College of Sciences

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Center for the Study of Systems Biology

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Publication Search Results

Now showing 1 - 10 of 19

Role of a Ribosome-Associated Chaperone in Viability, Prion Propagation, and Stress Memory

(Georgia Institute of Technology, 2022-07-25) Jay Garcia, Lina Manuela

Misfolding occurs when proteins fail to fold into their proper functional state. Normally, most misfolded proteins refold or are targeted for degradation through a special network of chaperones in order to maintain cellular proteostasis. However, in some cases, misfolded proteins can form ordered fibrous aggregates called amyloids. Amyloids are generally associated with aging, with neurological disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, and with a variety of other disorders, such as Type II diabetes and atherosclerosis. Yeast transmissible amyloids, termed yeast prions, are self-perpetuating heritable protein isoforms. Prion propagation in yeast is controlled by the chaperone machinery which includes Hsp104, Hsp70, and Hsp40 proteins. In this work, the yeast Saccharomyces cerevisiae is employed as a model to understand the molecular basis of how chaperones regulate the formation and propagation of amyloids. Here, I focused on the ribosome-associated chaperone Ssb, a member of the Hsp70 family, encoded by two genes (SSB1 and SSB2). My data indicate that during heat shock, Ssb is released from the ribosome and localized to the cytosol, interfering with Ssa, another Hsp70 protein, and impairing propagation of the prion [PSI+]. Attachment of Ssb1 to the activation domain of the protein Gal4, which contains a strong nuclear localization signal (AD-Ssb1), causes re-localization of Ssb to the nucleus. This leads to cytotoxicity and interference with propagation of [PSI+], [URE3], and [PIN+], prion forms of the Sup35, Ure2 and Rnq1 proteins, respectively. Moreover, relocation of the modified Ssb (AD-Ssb1) to the nucleus affects the function of Ssa directly or through a co-chaperone that is important for Ssa to function. I also found that deletion of ZUO1 (coding for the Hsp40 cochaperone of Ssb) increases spontaneous formation of the prion [URE3]. Moreover, in the absence of Ssb, mitotic stability of the prion forms of Ure2 ([URE3]) and Lsb2 ([LSB+]) is increased, while normally non-heritable mnemon aggregates of Ste18, [STE+] become heritable. De novo formation of [LSB+] and [STE+] is also increased in the absence of Ssb, especially during heat shock, which leads to massive accumulation of the [LSB+] prions. Overall, these results indicate that Ssb is a general anti-prion regulator, whose impact is not restricted only to the [PSI+] prion. In combination with the ribosome-associated chaperone complex (RAC), Ssb acts as a general modulator of cytosolic amyloid aggregation and can, directly or indirectly, repress prion generation and cure prions after they arise, counteracting prion toxicity. To further explore interactions among chaperones, prions and ribosomal machinery, I have constructed a yeast [PSI+] strain containing a deletion of all chromosomal rRNA genes (rdn1Δ), whose viability is dependent on a multicopy rRNA coding plasmid. This work expands the current knowledge of the role ribosome apparatus and ribosome-associated chaperones in heritable protein aggregation.
Investigating the aggregation of Alzheimer’s disease-associated proteins in S. cerevisiae

(Georgia Institute of Technology, 2019-05) Denniss, Julia Marie

Alzheimer’s disease (AD) is the most common type of dementia and is associated with roughly 500,000 new cases each year (2). AD is associated with the aggregation of two proteins in the brain, A-beta peptide (Aβ) and microtubule associated protein tau (MAPT). Aβ and MAPT are capable of adopting a cross-β fibrous protein structure, which can be reproduced and spread via nucleated polymerization and are termed amyloids. Despite such a broad biological impact of amyloids and prions, the mechanism of their initial formation in vivo remains a mystery. In this thesis, I will investigate proteins associated with AD, and the properties of these proteins that control their aggregation. Recent research has indicated that the U1 small nuclear ribonuclear protein 70 (U1-70k) can form detergent-insoluble aggregates in a manner specific to Alzheimer’s disease. U1-70k is strongly correlated with Aβ and tau, both proteins known to play a highly important role in the Alzheimer’s disease cascade and plaque formation. It has been shown that misfolded forms of U1-70k can sequester natively folded U1-70k proteins and cause them to form insoluble aggregates, a characteristic of amyloids (8). The mechanism behind this conversion remains elusive, however. Our research focuses on determining which domains and combinations of domains of the U1-70k protein are necessary for aggregation, and we also examine its interactions with Aβ. Through plasmid construction, expression, and observation under fluorescence microscopy (FM), we demonstrate that the N(1-99) domain alone cannot induce aggregation, but the C(182-437) domain, combined N and M domains, and M(100-181) domain are capable of inducing aggregation. Further SDD-AGE and Western blot analyses indicate that the aggregates formed by the C(182-437) domain are detergent-insoluble, while those formed by the N and M domains as well as the M(100-181) domain alone are detergent-soluble. This leads us to hypothesize that the aggregates formed by the M domain are reversible stress granules. Furthermore, the N and M domains also co-aggregate with Aβ, though the C(182-437) domain does not. We also examine tau’s suitability as a model in yeast for protein interactions and find that its aggregation is transformant-specific and cannot be cured by Hsp104, a heat shock protein found in yeast cells. We find that wild-type repeat domains of tau, the 244-372 amino acid region, aggregates are detergent-soluble.
Determining the effect of SSB on [LSB+] prion-based stress memory

(Georgia Institute of Technology, 2019-05) Faber, Quincy

Prions are self-perpetuating protein isoforms, that are usually based on ordered fibrous protein aggregates (amyloids), cause disease in humans and control non-Mendelian heritable traits in yeast. Formation and loss of yeast prions are modulated by environmental and physiological conditions, including heat stress. [LSB+], a metastable prion generated by the cytoskeleton-associated protein Lsb2 and influencing aggregation of other proteins, is induced by heat stress and persists in a fraction of yeast cells for a number of cell generations after stress, thus generating a cellular memory of stress. Chaperone proteins control protein folding, play an important role in adaptation during stress conditions, and are involved in prion formation and propagation. Ssb is member of the Hsp70 chaperone family and is normally associated with translating ribosomes. Previous studies in our lab indicated that Hsp70-Ssb has an anti-prion effect. We show that the formation and mitotic stability of the [LSB+] prion are greatly increased in the absence of Ssb. This links the ribosome-associated chaperone machinery to the cellular memory of stress.
Prion nucleation and propagation by mammalian amyloidogenic proteins in yeast

(Georgia Institute of Technology, 2018-04-17) Chandramowlishwaran, Pavithra

Cross-β fibrous protein polymers or “amyloids” are associated with a variety of human and animal diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) and are suspected to possess transmissible (prion) properties. However, the molecular mechanisms of amyloid formation and propagation are difficult to investigate in vivo due to complexity of the human organism. While evolutionarily distant from humans, yeast cells carry transmissible amyloids (yeast prions) that can be detected phenotypically. The objectives of the work presented in this dissertation were to understand the molecular mechanisms of initial prion nucleation and propagation by mammalian proteins in yeast. Our model employed chimeric constructs, containing the mammalian amyloidogenic proteins (or domains) fused to various fragments of the yeast prion protein Sup35. Phenotypic and biochemical detection assays, previously developed for the Sup35 prion, enabled us to detect prion nucleation and propagation by mammalian proteins. We have demonstrated that several non-Q/N rich, mammalian amyloidogenic proteins, nucleated a prion in yeast in the absence of pre-existing prions. Sequence alterations antagonizing or enhancing amyloidogenicity of human Aβ (associated with AD) and mouse PrP (associated with prion diseases) respectively antagonized or enhanced nucleation of a yeast prion by these proteins. Mutational dissection of Aβ identified sequences and chemicals that influence initial amyloid nucleation. We have also shown that Aβ and microtubule-associated binding protein tau that is also associated with AD, could propagate a prion state on their own or after transfection with in vitro generated amyloid seeds, in yeast. Aβ- and tau-based chimeric constructs formed distinct variants (“strains”) in the yeast cell. Our data show that prion properties of mammalian proteins detected in the yeast assays correspond with those found in mammals or in vitro, thus making yeast a powerful model for deciphering molecular foundations of amyloid/prion diseases.
Role of asymmetric segregation and the ribosome associated complex in prion formation and propagation

(Georgia Institute of Technology, 2018-04-06) Howie, Rebecca Leigh

Self-perpetuating transmissible protein aggregates—prions—are implicated in mammalian diseases and control phenotypically detectable traits in yeast. Yeast heat shock-induced chaperone proteins counteract stress-induced aggregation but also control prion propagation. Heat-damaged proteins that are not disaggregated by chaperones are cleared from daughter cells via mother-specific asymmetric segregation in cell divisions following heat shock. Heat shock-mediated destabilization of [PSI+], a prion isoform of the yeast translation termination factor Sup35, was previously shown to coincide with the imbalance between the Hsp104 and Ssa chaperones. The ribosome associated complex chaperone Ssb has previously been shown to antagonize the function of Ssa in prion propagation. The objective of this work was to better understand prion curing and formation in yeast, and specifically to understand the roles of asymmetric segregation and the ribosome associated complex. We show that cells lacking Sir2, which is responsible for asymmetric segregation of heat-damaged proteins, are impaired in the heat shock-mediated destabilization of [PSI+], and that Sup35 aggregates co-localize with aggregates of heat-damaged proteins. These results support the role of asymmetric segregation in prion destabilization. We then show that depletion of Ssb decreases heat shock-mediated destabilization of [PSI+], while disruption of a co-chaperone complex mediating the binding of Ssb to the ribosome increases prion loss. Ssb is shown to relocate from the ribosome to the cytosol during heat stress. These data support the role of Ssb, a stress non-inducible protein, in prion curing during stress and further implicate chaperone imbalance in prion curing. Lastly, we demonstrate that increased aggregation due to disruption of Ssb or the ribosome-associated complex increases formation of various prions, especially during stress, establishing these as anti-prion components that are necessary for both curing and protection against prion formation.
Discovery, characterization and mechanism of RNA and CDNA-mediated DNA double-strand break repair

(Georgia Institute of Technology, 2017-04-06) Keskin, Havva

A double-strand break (DSB) is one of the most deleterious DNA lesions and its repair is crucial for genome stability. Even if a single DSB is not repaired precisely, this could cause mutations, chromosomal rearrangements, cell death, and apoptosis. The safest mechanism to repair a DSB is homologous recombination (HR). HR requires an identical or nearly identical DNA template, such as a sister chromatid or a homologous chromosome to retrieve the missing genetic information and accomplish error-free repair. In special cases, HR can occur between RNA molecules, such as RNA molecules in RNA viruses. However, very little is known about RNA-DNA HR. Previously, it was demonstrated that synthetic RNA-containing molecules can serve as templates for repairing defective or broken homologous chromosomal DNA in yeast, human and bacterial cells, but it remained unclear whether cellular RNA transcripts can recombine with genomic DNA. Here, we investigated whether yeast cells can use transcript RNA as a template to repair a chromosomal DSB either directly or indirectly, if the RNA is converted first into a DNA copy, cDNA. We developed a system to detect HR between chromosomal DNA and transcript RNA in budding yeast, Saccharomyces cerevisiae. We focused on repair of a chromosomal DSB occurring either in a homologous but remote locus (trans) or in the same transcript-generating locus (cis) in yeast. We proved that transcript RNA can repair a DSB indirectly, via cDNA. Moreover, we found that cDNA repair is much more frequent in the trans than in the cis system. Interestingly, in the absence of Ribonuclease H1 and H2 (RNases H1 and H2), we could detect DSB repair even in conditions that strongly inhibit cDNA formation, suggesting direct DSB repair by transcript RNA. In contrast to DSB repair by cDNA, the direct DSB repair by transcript RNA is more efficient in the cis than in the trans system, despite the higher abundance of the transcript in the trans system. These results suggest that the vicinity of the transcript RNA to the break site in the cis system may facilitate DSB repair. DSB repair by transcript RNA in cis is promoted by the HR protein Rad52 but not Rad51, in agreement with the demonstration that the yeast and human Rad52 proteins efficiently catalyze annealing of RNA to a DSB-like DNA end in vitro. We also showed that yeast cells expressing hypomorphic mutants of RNase H2, which correspond to the human RNase H2 mutants that are associated with the neuroimmunological disease, Aicardi Goutieres (AGS) syndrome, have increased frequency of DSB repair by cDNA, significantly higher than in wild-type RNase H2 cells. In addition, we showed that in contrast to DSB repair by single strand DNA (ssDNA) oligonucleotides (oligos), RNA templated DSB repair is not dependent on factors that are major players in DNA end resection. This result could be explained by a mechanism in which transcript RNA repairs a DSB in conditions of limited end resection via an inverse strand exchange reaction. Our study provides proof and initial characterization of a new mechanism of DNA repair and HR mediated by RNA in yeast, and unravels novel aspects in the complex relationship between RNA and DNA in genome stability.
Roles of protein sequence and cell environment in cross-species prion transmission and amyloid interference

(Georgia Institute of Technology, 2014-07-08) Bruce, Kathryn Lyn

Proteinaceous infectious particles, termed 'prions' are self-perpetuating protein isoforms that transmit neurodegenerative diseases in mammals and phenotypic traits in yeast. Each conformational variant of a prion protein is faithfully propagated to a homologous protein in the same cell environment. However, a reduction in the efficiency of prion transmission between different species is often observed and is termed "species barrier". Prion transmission to a heterologous protein may, in some cases, permanently change the structure of the prion variant, and divergent proteins may interfere with prion propagation in a species-specific manner. To identify the importance of both protein sequence and the cell environment on prion interference and cross-species transmission, we employed heterologous Sup35 proteins from three Saccharomyces sensu stricto species: Saccharomyces cerevisiae (Sc), Saccharomyces paradoxus (Sp), and Saccharomyces bayanus (Sb). We performed our experiments in two different cell environments (Sc and Sp). Our data show that Sup35 from one species can form a prion in another, and we employed a transfection procedure to perform cross-species transfer of the prion. Using a shuffle procedure, we demonstrate that the specificity of prion transmission is determined by the protein itself rather than the cell environment. Interestingly, we noted that variant-specific prion patterns can be altered irreversibly during cross-species transmission through S. bayanus module II. We further show that prion interference does not always correlate with cross-species prion transmission, and the identity of particular regions or even a specific amino acid, rather than the overall level of PrD homology is crucial for determining cross-species transmission and interference. Lastly we provide evidence to suggest that prion interference is specific to the cell environment.
Studies of genetic factors modulating polyglutamine toxicity in the yeast model

(Georgia Institute of Technology, 2011-09-28) Gong, He

Polyglutamine-expanded fragments, derived from the human huntingtin protein, are aggregation-prone and toxic in yeast cells, bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline-rich region targets polyglutamine aggregates to the large perinuclear deposit (aggresome). Aggresome targeting ameliorates polyglutamine cytotoxicity in the presence of the prion form of Rnq1 protein, however, aggresome-forming construct remains toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). Disomy by chromosome II partly ameliorates polyglutamine toxicity in the strains containing Sup35 prion. The chromosome II gene, coding for another release factor, and interaction partner of Sup35, named Sup45 (eRF1), is responsible for amelioration of toxicity. Plasmid-mediated overproduction of Sup45, or expression of the Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into prion aggregates, also ameliorate polyglutamine toxicity. Protein analysis indicates that polyglutamines alter aggregation patterns of the Sup35 prion and promote aggregation of Sup45, while excess Sup45 counteracts these effects. In the absence of Sup35 prion, disomy by chromosome II is still able to decrease polyglutamine toxicity. However, SUP45 is no longer the gene responsible for such an effect. Taken together with the finding that the presence of both the Rnq1 prion and the Sup35 prion has an additive effect on polyQ toxicity, one gene or few genes on chromosome II are able to ameliorate polyQ toxicity through a SUP45-independent pathway. The identification of such a gene is currently ongoing. Monosomy by chromosome VIII in diploid heterozygous by AQT (Anti-polyQ Toxicity mutants that are disomic by chromosome II) counteracted the effect of AQT. Similarly, deletion of the arg4 gene in chromosome VIII in AQT haploid was able to eliminate the AQT effect. Moreover, analysis of genes involved in the arginine and polyamine synthesis indicated that loss of genes in later stages of arginine biosynthesis causes increase of polyglutamine toxicity. Deletion of genes arg1, arg4, arg8 (arginine pathway) and spe1 (polyamine pathway) all suppressed the Sup35 prion phenotype expression in the nonsense suppression system. Further analysis regarding the mechanisms behind those effects is needed. Our data uncover the mechanisms by which genetic and epigenetic factors may influence polyglutamine toxicity, and demonstrate that one and the same type of polyglutamine deposits could be cytoprotective or cytotoxic, depending on the prion composition of a eukaryotic cell.
Development of the new yeast-based assays for prion properties

(Georgia Institute of Technology, 2011-08-29) Sun, Meng

Prion is an infectious isoform of a normal cellular protein which is capable of converting the non-prion form of the same protein into the alternative prion form. Mammalian prion protein PrP is responsible for prion formation in mammals, causing a series of fatal and incurable prion diseases. (1) We constructed, for the first time, a two-component system to phenotypically monitor the conformational status of PrP in the yeast cells. In this system, the prion domain of Sup35 (Sup35N) was fused to PrP90-230, and the initial formation of the PrPSc-like conformation stimulated prion formation of Sup35N, which in turn converted soluble Sup35 into the prion isoform, leading to a detectable phenotype. Prion-like properties of PrP were studied in this novel yeast model system. Additionally, we employed this system to study amyloidogenic protein Aβ42 aggregation in the yeast model. It has been suggested that the ability to form transmissible amyloids (prions) is widespread among yeast proteins and is likely intrinsic to proteins from other organisms. However, the distribution of yeast prions in natural conditions is not yet clear, which prevents us from understanding the relationship between prions and their adaptive roles in various environmental conditions. (2) We modified and developed sequence and phenotype-independent approaches for prion detection and monitoring. We employed these approaches for prion-profiling among yeast strains of various origins. (3) Lastly, we found a prion-like state [MCS+] causing nonsense suppression in the absence of the Sup35 prion domain. Our results suggested that [MCS+] is determined by both a prion factor and a nuclear factor. The prion-related properties of [MCS+] were studied by genetic and biochemical approaches.
Genetic and physical interaction of Sgt2 protein with prion-chaperone machinery

(Georgia Institute of Technology, 2011-08-10) Pan, Tao

The word "Prion" refers to self-perpetuating protein aggregates that cause neurodegenerative diseases in mammals. It is a protein isoform that has undergone a conformational change which converts the normal form of the protein into the infectious form with the same amino acid sequence. Yeast [PSI+] prion is the prion isoform of Sup35 protein, a translation termination factor eRF3. It has been suggested that prion [PSI+] is controlled by the ensemble of chaperones with Hsp104 playing the major role. The previous work performed in the Chernoffs lab showed that the defective GET pathway caused by get led to the defect in [PSI+] curing by excess Hsp104. The GET pathway is a system responsible for transporting newly synthesized TA-protein to the ER membrane, and the components which have been proven to be involved in this pathway include: Get1, Get2, Get3, Get4, Get5 and Sgt2. In this study we describe the mechanism underlying the effect of the defective GET pathway on [PSI+]. We demonstrate that Sgt2, one of the components of GET pathway, interacts with Sup35 in both [PSI+] and [psi-] strains through its prion domain. Overproduction of Sgt2 and Hsp70-Ssa is triggered by the defective GET pathway and leads to the protection of [PSI+] aggregates from curing by excess Hsp104. We show that the direct interaction between Sgt2 and Hsp70-Ssa is not required for this protective effect.